The use of modifiers of the biological response for treating cancer mainly combined with current therapies to enhance the benefit of treatment has been recently reported (US 2004/0101511). On the other hand, the use of CpG sequences, the agonist of the Toll-like receptor 9 (TLR9), has been developed as new drugs for treating, controlling and preventing cancer as parts of multiple treatment indications, i.e. non-small cells lung cancer, melanoma and renal carcinoma (Klinman D. M., et al, (2004) Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat Rev Immunol. 4:249-258). A Toll-like receptor 7 (TLR7) agonist is currently tested in phase I clinical trials for activating the immune system, with promising results as a new drug for treating melanoma and other tumors (Dudek A. Z., et al (2005) ASCO Annual Meeting). The previously mentioned agonists of TLRs 7 and 9 are also being evaluated in viral infections, based on their capacity to promote an effective immune response in the host. Besides, the named Heat shock proteins (Hsp) that bind to the TLR4, have been developed and manufactured as a fusion protein to the human papillomavirus (HPV) E7 oncoprotein. This new immunotherapeutic approach is also known as therapeutic vaccines, with (Chu N. R. et al., (2000) Immunotherapy of a human papillomavirus (HPV) type 16 E7-expressing tumour by administration of fusion protein comprising Mycobacterium bovis bacille Calmette-Guerin (BCG) hsp65 and HPV16 E7. Clin Exp Immunol 121:216-225). a wide perspective for treating human papillomavirus-related diseases. Toll-like receptors are receptor molecules present in cells of the immune system, recognizing pathogen-associated molecular patterns, like LPS, lipoteichoic acid, unmethylated CpG sequences and viral double- and single-stranded RNA. The recognition of the invading pathogen by TLRs helps the immune system to direct a balanced Th1/Th2 immune response to efficiently eradicate the infection out of the organism. The use of TLRs agonists as drugs to treat cancer is based on the activating the innate and adaptive immune systems, by activating a Th1 immune response mediated by type I interferons (e.g. α & β IFNs) and interleukin 12 (IL-12) as the main mechanism. Therefore, a highly specific and sustained immune response is achieved (Switaj T., Jalili A., et al., (2004) CpG Immunostimulatory oligodeoxynucleotide 1826 enhances antitumor effect of interleukin 12 gene-modified tumor vaccine in a melanoma model in mice. Clinical Cancer Research, Vol. 10:4165-4175). This dual activation of the immune system contrasts with several other immunotherapeutic approaches which are unable to generate a sustained effect in the adaptive immune response and also unspecifically activating the innate immune system with subsequent undesired effects (Speiser D. E, et al. (2005) Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. The Journal of Clinical Invest. Vol. 115 (3)).
Dendritic cells (DCs) are professional antigen presenting cells linking innate and adaptive immune responses through cell-to-cell interactions and cytokine production. DCs are classified in myeloid or lymphoid attending to their origin, based on differential expression of a series of surface molecular markers and also TLRs. Lymphoid DCs, also known as plasmacytoid DCs, are the main source of type I interferons. Considering these properties, DCs have been manipulated as promising cellular adjuvants to develop therapeutic vaccines against cancer and chronic viral infections (Santini S. M., et al (2003) A new type I IFN-mediated pathway for rapid differentiation of monocytes into highly active dendritic cells. Stem Cells, 21:357-362). However, this is a very expensive and difficult technique, with other more practical and less expensive therapeutic strategies under development (Van Epps H. L. (2005) New hope for tumor vaccines. The Journal of Experimental Medicine, Vol. 202:1615).
Originally described by their antiviral activity, type I interferons (α,β IFNs) have been recently shown to exert important effects over the immune system, promoting cellular and humoral immune responses through their adjuvant effects on DCs (Bogdan, C. (2000) The function of type I interferons in antimicrobial immunity. Curr, Opin Immunol. 12:419-424). Recent works have shed lights on the critical role of endogenous type I interferons in processes mediating regression of a highly immunogenic syngenic murine sarcoma, and protecting the host against the occurrence of primary carcinogenic tumor (Gavin P. Dunn, et al. (2005) A critical function for type I interferons in cancer immunoediting. Nature Immunology, June 12). Resides, IFN-α plays an important role in initiating the antiviral T-lymphocyte response through direct activation of CD4+ or CD8+ T lymphocytes in viral infections like Influenza (Fonteneau J. F, et al. (2003) Activation of influenza virus-specific CD+4 and CD+8 T cells: a new role for plasmacytoid dendritic cells in adaptive immunity. Immunobiology, 101: 3520-3526).
Hoess (WO 95/05393) relates in his invention substances binding LPS with high affinity, useful for preventing or treating infections like gram-positive or gram-negative bacteria-mediated sepsis, bacterial infections in general and fungal infections. Such substances are LPS-binding peptides bearing an endotoxin-binding domain (Hoess A., et al, (1993) Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5 A° resolution. The EMBO J. 12:3351-3356). The crystal structure of the original Limulus anti-LPS factor (LALF) protein reveals a loop similar to polymyxin B, positively charged, amphipathic and containing exposed hydrophobic and aromatic residues. Based on this principle, he documented the capacity of sequences corresponding to aminoacids 31 to 52 on the LALF protein to bind and neutralize the effects associated to heparin, like anti-coagulation, angiogenesis and inhibition of endothelial and tumor cells' proliferation. However, there are no experimental data supporting this statement in the patent mentioned above. In fact, claims granted referred to a device for removing LPS in solution, wherein said device comprises peptides immobilized in a solid support (U.S. Pat. No. 6,384,188).
On the other hand, Vallespi (U.S. Pat. No. 6,191,114) relates in her invention the antiviral effect of the LALF31-52 peptide on Hep-2 and MDBK cells, mediated by the production of α and γ interferons, also relating her invention to the use of this peptide for treating viral infections and immunosuppression-related disorders. Moreover, the same author has demonstrated the anti-infection effect of this peptide in animal models of sepsis (Vallespi M. G., et all (2003) A Limulus anti-LPS factor-derived peptide modulates cytokine gene expression and promotes resolution of bacterial acute infection in mice. International Immunopharmacology, 3:247-256).
There are a number of therapies directed against cancer, including chemotherapy, radiations and gene therapy. Toxicity is one major disadvantage of all these therapies, with high doses been administered for prolonged periods of time to finally achieve some beneficial therapeutic effect. Therefore, there is still required the development of new drugs for gaining more effective treatments.
Based on the essential role of the immune system to detect and direct an efficient response against tumors, drugs designed to activate the host innate and adaptative defense mechanisms can become powerful tools for cancer therapeutics.
There are no previous amino acid substitutions described for the sequence HYRIKPTFRRLKWKYKGKFW (SEQ ID NO: 13) of the LALF protein, eliminating its LPS-binding capacity and enhancing the immunomodulatory effect, also conferring anti-tumoral effects in vivo against several tumors.